The field of analytical chemistry and biochemistry is concerned with the measurement of composition, amounts and properties of substances, such as solutions of chemicals, biochemical substances, or other analytes. An important biochemical project that has been completed in the past years is the human genome project. This project has brought up a new business field, pharmacogenomics, which has the goal to understand biological functions and to influence such functions by specific interventions with pharmaceutical substances. By comparing protein-expression after drug treatment with protein expression in untreated condition, it is possible to relate the observed changes to the effects or function of the drug. Recent works have shown that there is a lack of correlation between transcriptional profiles and actual protein levels in cells. Protein analysis has therefore become indispensable and complementary to genomic analysis in order to obtain an accurate picture of cellular function and metabolism.
Methods and apparatus have been developed to enable detection or identification of analytes in smaller and smaller amounts or under more useful conditions. In particular, protein analysis requires speedy execution and high separation efficiency if applied as a screening method for various cell and tissue types. The established method for protein expression monitoring is gel electrophoresis on polyacrylamide (PAGE). While this is efficient as a preparative method, it is by far too slow for screening applications. It requires a lot of time involvement of an operator, and often the separated spots have to be cut out manually from these preparations so that they can subsequently be analyzed in a Maldi-TOF-MS system (Maldi: matrix assisted laser desorption ionization; TOF:time-of-flight; MS: mass spectrometry).
Often it is difficult to achieve enough separation or peak capacity in order to detect and identify all individual fragments of a sample in just one run. This limitation usually is a reason to use two-dimensional separation devices wherein a hybrid of two separation mechanisms is formed. Examples for such two-dimensional separations are: ion exchange/reversed phase liquid chromatography, or isoelectric focusing (IEF)/SDS-PAGE, which is called two-dimensional gel electrophoresis (2D-GE). Regarding state of the art, it is referred to the article, G. L. Corthals et al.: “The dynamic range of protein expression: A challenge for proteomic research”, Electrophoresis 2000, 21/6, pages 1104–1115; and V. C. Wasinger et al.: “Proteomic tools for biomedicine”, Journal of Chromatography B, 771 (2002), pages 33–48.
In the pharmaceutical industry, high throughput screening is often required. In order to screen for statistically relevant parameters, a large number of analysis steps are performed in parallel. High throughput screening requires automation, speed and reliable operation. In this approach often all the 2D-GE platforms cannot fulfill the requirements.
Several attempts have been made to create an automated two-dimensional separation arrangement. In one approach, liquid chromatography is combined with capillary electrophoresis. In another approach, disclosed in EP-A 977030, a two-dimensional electrophoresis separation device is implemented on a microchip.
The mentioned devices and methods have their primary applications in the field of proteomics. There is such a broad variety of proteins that a simple uni-dimensional separation quickly reaches its limits. Furthermore, the interesting proteins in a sample indicating specific disorders or diseases often are only present in small quantities, whereas there is a substantially larger portion of proteins in the sample which are not of particular interest. This has the consequence that small, but interesting peaks in the measuring results are concealed under tall peaks which are of no particular interest for the actual analysis, which in turn requires even more peak capacity to get clearer, more distinct readings.
Reliable results in the field of proteomics are so important that one even has to take the trouble of several-days measurements in order to produce a clear distinct two-dimensional pattern. It has been common to run a two-dimensional gel electrophoresis, cut out the corresponding spots to extract a clean population and then to run it on a Maldi-MS or even a sequencer, see also Mary F. Lopez: “Better approaches to finding the needle in a haystack: Optimizing proteome analysis through automation”, Electrophoresis, Vol. 21, Issue 6 (2000), Pages 1082–1093.